Discovery of sulfonamide-tethered isatin derivatives as novel anticancer agents and VEGFR-2 inhibitors

Abstract In this work, new isatin-based sulphonamides (6a-i, 11a-c, 12a-c) were designed and synthesised as potential dual VEGFR-2 and carbonic anhydrase inhibitors with anticancer activities. Firstly, all target isatins were examined for in vitro antitumor action on NCI-USA panel (58 tumour cell lines). Then, the most potent derivatives were examined for the potential CA inhibitory action towards the physiologically relevant hCA isoforms I, II, and tumour-linked hCA IX isoform, in addition, the VEGFR-2 inhibitory activity was evaluated. The target sulphonamides failed to inhibit the CA isoforms that could be attributable to the steric effect of the neighbouring methoxy group, whereas they displayed potent VEGFR-2 inhibitory effect. Following that, isatins 11b and 12b were tested for their influence on the cell cycle disturbance, and towards the apoptotic potential. Finally, detailed molecular modelling analyses, including docking and molecular dynamics, were carried out to assess the binding mode and stability of target isatins.


In vitro cytotoxicity
The cytotoxic efficacy of the target compounds against T47D cells was determined, using the SRB colorimetric technique, as previously reported. Briefly, exponentially growing cells were trypsinized, counted and seeded at the appropriate densities (5000 cells/100 μL/ well) into 96-well microtiter plates. Cells were incubated in a humidified atmosphere at 37 °C for 24 h.
Then, the cells were exposed to the tested compounds at the desired concentrations (0.01, 0.1, 1, 10, and 100 μM) or to 1% dimethyl sulfoxide (DMSO) for 72 h. At the end of the treatment period, the media were removed, and the cells were fixed with 10% trichloroacetic acid at 4 °C for 1 h. Following, the cells were washed with tap water four times and incubated with SRB 0.4% for 30 min. Excess dye was removed by washing repeatedly with 1% (vol/vol) acetic acid.
The protein-bound dye was dissolved in 10 mM Tris base solution for (optical density) OD determination at 510 nm using a Spectra Max plus Microplate Reader (Molecular Devices, CA).
Cell viability was expressed relative to the untreated control cells 2, 3 .

VEGFR-2 inhibition assay
This assay was performed according to the reported method 4,5

Carbonic anhydrase I, II, and IX inhibition studies
The CO2 hydration actions catalyzed by carbonic anhydrase for targeted compounds herein the cells were stained with propidium iodide for 30 min. Cell cycle analysis and apoptosis were conducted using a FACS Calibur flow cytometer as shown in previous studies 8,9 . Every experiment has been carried out three times.

Annexin V-FITC apoptosis assay
Cells were incubated with the tested compounds 11b and 12b for 24h, trypsinized and washed with cold phosphate-buffer saline (PBS). Cells were then stained with Annexin V-FITC and PI in binding buffer for 15 min at room temperature in the dark. The samples were analyzed using the flow cytometer 8,9 .

MD simulation
GROMACS 2021 was used to run molecular dynamics (MD) simulations on the best docking poses 10 . The CHARMM General Force Field (CGenFF) server was utilized to generate the ligands topology, whereas the input files for the MD calculations were created applying the CHARMM force field parameters for protein 11 . They were immersed in a TIP3P water model cubic box with 10 padding and neutralized by adding Na + and Clions. For long-range electrostatic interactions and Non-bonded interactions were treated with a 12 ˚A cutoff distance and the neighbor searching list were buffered with the Verlet cutoff-scheme 12 and the long-range electrostatic interactions were treated with the particle mesh Ewald (PME) method 13 .
CHARMM36 forcefield was applied on the protein-ligand complex 14 . Prior to production simulation, energy minimization of the system was carried out by using steepest descent algorithm (5000 steps). The complex was then equilibrated for stabilizing its temperature and pressure by subjecting it to NVT and NPT ensemble and simulating for 125 ps at 300.15 K temperature using 400 kJ mol −1 nm −2 and 40 kJ mol −1 nm −2 positional restraints on the backbone and side chains, respectively. Finally, the complex is subjected to production simulation run for 100 ns in NPT ensemble at 300.15 K and 1 bar. To maintain the temperature Nose-Hoover thermostat was used 15 and similarly for maintaining the pressure Parrinello-Rahman barostat was used 16 . LINCS algorithm was used for constraining H-bonds 17 . The V-rescale thermostat at 300 K with a coupling constant of 1 ps was used. The trajectories were stored every 2 ps.
Simulations of 100 ns in NPT assembly were performed for the production stage. GROMACS utilities were used for the analysis of the MD simulations 18